MicroRNA Genes and Their Functions Project Summary/Abstract The role that RNA molecules play during eukaryotic gene expression has moved to center stage. Land plants, mammals, and other bilateral animals each have from ~100 to >400 genes that produce ~22-nt noncoding RNAs, called microRNAs (miRNAs). MicroRNAs pair to the mRNAs of protein-coding genes to direct the posttranscriptional repression of these messages. Because a single mammalian miRNA can target hundreds of messages, miRNAs have a widespread impact on mRNA repression and evolution. This proposal focuses on the genomics and functional genomics of this newly identified class of genes, with the broad, long-term objective of understanding the roles of RNA in regulating gene expression. The specific aims are: 1) to identify additional miRNA genes, 2) to improve and expand miRNA target identification, and 3) to determine the molecular consequences of miRNA-mediated repression. Experiments of Aim #1 will use high-throughput sequencing to identify many miRNA genes, including previously unrecognized human genes, substantially increasing (and correcting) annotations of this gene class. Gene discovery will also be extended to basal animals, including sponge and cnideria, to shed light on the evolutionary origins of this mode of gene regulation. Experiments of Aim #2 will use molecular and comparative genomic approaches to provide a more precise and comprehensive list of predicted targets a resource for all biologists, and of particular value for those concerned with eukaryotic gene regulation, development, and disease. Experiments of Aim #3 will determine the extent to which miRNA-mediated repression can be discerned from reduced transcript accumulation. They also will likely identify many new targets beyond those currently detected on mRNA expression arrays and thereby provide large, quantitative datasets needed for improving and expanding target predictions. Because more than a third of the human genes have been under selective pressure to maintain their pairing to a set of broadly conserved miRNAs, it is no surprise that miRNAs have already been shown to play important roles in mammalian development, viral infection, and cancer. Knowing the miRNA genes, their likely targets, and the molecular consequences of their action will be useful for determining additional functions of miRNAs and learning how their dysfunction can contribute to human disease. Public Health Relevance: The experiments of this proposal focus on short snippets of RNA called microRNAs, which can collaborate with the proteins of the cell to determine how much protein is produced from individual genes, including many genes implicated in cancer and other diseases. The goals are to find microRNAs that had gone unrecognized, to learn how best to predict which genes the microRNAs regulate, and to determine the magnitude of their effects on protein output. Achieving these goals will shed light on why microRNAs are needed for normal growth and development, and how their dysfunction can contribute to human diseases.